Bottom Line:
Here, we describe a novel system that allows verification of phenotypic changes attributed to expression of genes from the HAC without a transfection step.We demonstrated that a single copy of tTA(VP64) carrying four tandem repeats of the VP16 domain constitutively expressed from the HAC is capable to generate chromatin changes in the HAC kinetochore that are not compatible with its function.To adopt the alphoid(tetO)-HAC for routine gene function studies, we constructed a new TAR-BRV- tTA(VP64) cloning vector that allows a selective isolation of a gene of interest from genomic DNA in yeast followed by its direct transfer to bacterial cells and subsequent loading into the loxP site of the alphoid(tetO)-HAC in hamster CHO cells from where the HAC may be MMCT-transferred to the recipient human cells.

Figure 1: Loading of the tTAVP64-containing construct into the alphoidtetO-HAC propagated in human HPRT-minus HT1080 cells. (a) Diagram of the construct tetR-VP64-IRES-DsRed2 used in this study. The construct contains a 3′ HPRT sequence and loxP site, the tTAVP64 (tetR with the VP64 carrying four copies of VP16 activation domain) that is co-transcribed with the DsRed2 transgene under the same CAG promoter. The cHS4 insulator (54) flanks the expressing cassette from both sides. (A similar construct, tetR*-VP16-IRES-DsRed2, carrying one copy of the VP16 domain is described in Supplementary Figure S2). Both constructs were inserted into the loxP site of the alphoidtetO-HAC propagated in HPRT-deficient HT1080 cells. (b) Loading of the vector into the loxP site of the HAC is accompanied by reconstitution of the HPRT gene allowing cell selection on HAT medium. (c) A map of the resulting transgene cluster in the HAC containing TK and Hygro genes, the DsRed2 color marker and the reconstructed HPRT gene. Arrows indicate direction of transcription of the transgenes and 30–40 copies of BS incorporated in the alphoidtetO-HAC. (d) Lanes 1, 2 and 3 correspond to PCR products obtained with the genomic DNA isolated from HAC-containing clones of HT1080 cells using specific primers for the HPRT gene. The PCR products were sequenced and confirmed reconstitution of the HPRT gene. M-ladder marker. (e–g) FISH analysis of the HAC-containing HT1080 clone. Chromosomal DNA was counterstained with DAPI (blue) (e). The HAC was visualized using a BAC32–2-mer(tetO) probe (red) (f and g).

Mentions:
In this study, two transcriptional transactivator tTA -containing vectors, tetR-VP64-IRES-DsRed2 (Figure 1a; Supplementary Figure S1) and tetR*-VP16-IRES-DsRed2 (Supplementary Figure S2a) were constructed (see Materials and Methods for details) and then inserted into the loxP site of the alphoidtetO-HAC in HPRT-deficient human HT1080 cells (Figure 1b) in order to determine the consequences of a single copy tTA expression on HAC stability. The tetR-VP64-IRES-DsRed2 vector contains the tTAVP64 sequence (four tandem repeats of the minimal VP16 domain DALDDFDLDML fused with TetR), the color marker DsRed2 and the 3'-end HPRT sequence fragment. The tTAVP64 and DsRed2 sequences are transcribed from the same CAG promoter but are separated by an internal ribosome entry site (IRES) sequence (Figure 1a). Therefore, while the tetR-VP64-IRES-DsRed2 vector encodes a single tTAVP64/DsRed2 transcript it is translated into two proteins: tTAVP64 (which binds to the multiple tetO-sequences in the alphoid array of the HAC) and DsRed2 (which is translated from IRES at a lower efficiency). The cHS4 insulator flanks the whole cassette. The tetR*-VP16-IRES-DsRed2 vector is identical to tetR-VP64-IRES-DsRed2 but contains only one copy of the VP16 domain. The tTAVP64 or tTAVP16 binding to tetO sequences is negatively regulated by doxycycline.

Figure 1: Loading of the tTAVP64-containing construct into the alphoidtetO-HAC propagated in human HPRT-minus HT1080 cells. (a) Diagram of the construct tetR-VP64-IRES-DsRed2 used in this study. The construct contains a 3′ HPRT sequence and loxP site, the tTAVP64 (tetR with the VP64 carrying four copies of VP16 activation domain) that is co-transcribed with the DsRed2 transgene under the same CAG promoter. The cHS4 insulator (54) flanks the expressing cassette from both sides. (A similar construct, tetR*-VP16-IRES-DsRed2, carrying one copy of the VP16 domain is described in Supplementary Figure S2). Both constructs were inserted into the loxP site of the alphoidtetO-HAC propagated in HPRT-deficient HT1080 cells. (b) Loading of the vector into the loxP site of the HAC is accompanied by reconstitution of the HPRT gene allowing cell selection on HAT medium. (c) A map of the resulting transgene cluster in the HAC containing TK and Hygro genes, the DsRed2 color marker and the reconstructed HPRT gene. Arrows indicate direction of transcription of the transgenes and 30–40 copies of BS incorporated in the alphoidtetO-HAC. (d) Lanes 1, 2 and 3 correspond to PCR products obtained with the genomic DNA isolated from HAC-containing clones of HT1080 cells using specific primers for the HPRT gene. The PCR products were sequenced and confirmed reconstitution of the HPRT gene. M-ladder marker. (e–g) FISH analysis of the HAC-containing HT1080 clone. Chromosomal DNA was counterstained with DAPI (blue) (e). The HAC was visualized using a BAC32–2-mer(tetO) probe (red) (f and g).

Mentions:
In this study, two transcriptional transactivator tTA -containing vectors, tetR-VP64-IRES-DsRed2 (Figure 1a; Supplementary Figure S1) and tetR*-VP16-IRES-DsRed2 (Supplementary Figure S2a) were constructed (see Materials and Methods for details) and then inserted into the loxP site of the alphoidtetO-HAC in HPRT-deficient human HT1080 cells (Figure 1b) in order to determine the consequences of a single copy tTA expression on HAC stability. The tetR-VP64-IRES-DsRed2 vector contains the tTAVP64 sequence (four tandem repeats of the minimal VP16 domain DALDDFDLDML fused with TetR), the color marker DsRed2 and the 3'-end HPRT sequence fragment. The tTAVP64 and DsRed2 sequences are transcribed from the same CAG promoter but are separated by an internal ribosome entry site (IRES) sequence (Figure 1a). Therefore, while the tetR-VP64-IRES-DsRed2 vector encodes a single tTAVP64/DsRed2 transcript it is translated into two proteins: tTAVP64 (which binds to the multiple tetO-sequences in the alphoid array of the HAC) and DsRed2 (which is translated from IRES at a lower efficiency). The cHS4 insulator flanks the whole cassette. The tetR*-VP16-IRES-DsRed2 vector is identical to tetR-VP64-IRES-DsRed2 but contains only one copy of the VP16 domain. The tTAVP64 or tTAVP16 binding to tetO sequences is negatively regulated by doxycycline.

Bottom Line:
Here, we describe a novel system that allows verification of phenotypic changes attributed to expression of genes from the HAC without a transfection step.We demonstrated that a single copy of tTA(VP64) carrying four tandem repeats of the VP16 domain constitutively expressed from the HAC is capable to generate chromatin changes in the HAC kinetochore that are not compatible with its function.To adopt the alphoid(tetO)-HAC for routine gene function studies, we constructed a new TAR-BRV- tTA(VP64) cloning vector that allows a selective isolation of a gene of interest from genomic DNA in yeast followed by its direct transfer to bacterial cells and subsequent loading into the loxP site of the alphoid(tetO)-HAC in hamster CHO cells from where the HAC may be MMCT-transferred to the recipient human cells.